This invention relates to a method of differentiating and accurately counting nucleated red cells ("NRBC")in a whole blood sample. In particular this invention relates to a method for the simultaneous differentiation and counting of NRBC and white blood cell ("WBC") sub-populations in a whole blood sample by the use of two light scattering parameters and fluorescence.
Events related to the onset of the anemia need to be carefully monitored. The hematology laboratory offers a set of routine or standard procedures relevant to the diagnosis of anemia. The most important of these procedures are the complete blood count (performed on an automated blood cell counter), blood smear morphology, and the reticulocyte production index. NRBC counts are conventionally determined by means of blood smear morphology. A stained blood smear is examined under the microscope and the NRBC are manually counted. In general, an NRBC concentration is reported as number of NRBC per 100 white blood cells ("WBC"). Normally, 200 WBC and the number of NRBC present in the same region on a patient blood smear are counted and the numbers are divided by 2 to express the NRBC concentration as the number of NRBC/100 WBC. The major drawback to this type of manual microscopic method is that it is very labor intensive, time-consuming, subjective and inaccurate due to poor statistics. Therefore, an accurate automated NRBC method has long been sought after by pathologists and laboratory technicians.
A major problem in automating a NRBC method for use on a clinical flow cytometer has been that since NRBC are rare events and RBC populations are so numerous, NRBC populations are not easily detected among the red blood cell ("RBC") population by either the differences in the cell's electrical resistivity (impedance measurements) or its light scattering characteristics (optical measurements). Although many attempts have been made to count NRBC among WBC populations, instead of among RBC population, these efforts have not generally been successful.
NRBC populations are not easily distinguished from WBC populations since NRBC do not form a well defined cluster among the WBC in the usual two dimensional space differentiation methods utilized on flow cytometers. One is usually not able to separate NRBC populations from the lymphocyte populations when the detected signals are viewed on the generally accepted, two-dimensional light scatter (forward vs. side) or light scatter vs. absorption, dot plots. The signals from the majority of the NRBC population is usually mixed in with the signals for RBC stroma and platelets ("PLT"), and the upper-end of NRBC cluster most often will extend into the space occupied by the lymphocyte population.
Automated clinical hematology instruments, such as the Technicon H*1.RTM., Coulter STK.RTM. S and Abbott Cell-Dyn.RTM. 3000 and 3500 only "flag" samples for the possible presence of NRBC if the sample dot plot shows increased noise signals below the lymphocyte cluster. This type of flagging very often produces false positive results since the elevated noise level could be due to PLT clumps, giant PLT or incompletely lysed RBC. In addition, it is extremely difficult to obtain accurate Total WBC and WBC Differential ("WBC/Diff") results on samples containing NRBC because of the interference. Additionally, blood smears of the flagged samples must be examined and counted under the microscope by a skilled technician to obtain accurate WBC differential and NRBC counts. This is a very labor-intensive and subjective process.
Recently, U.S. Patent No. 5,298,426, issued on Mar. 29, 1994, to Inami et al. This patent teaches a two-step method comprising the staining of WBC and NRBC by specific nuclear stains. In this patented method, a blood sample is first mixed with an acid hypotonic solution containing a fluorescent nuclear dye. Then, a solution comprising an alkaline salt buffer, to adjust pH and Osmolarity, is mixed with the sample/first reagent solution. This final solution is then loaded into a flow cytometer to detect and count NRBC along with other nucleated cells.
There are several reasons why the Inami et al. approach is not acceptable, especially for an automatable method. First, an acidic-hypotonic solution damages all cell membranes making all WBC leaky and therefore selective staining of NRBC nuclei by a nuclear stain is not possible. There are no known dyes which stain only NRBC nuclei and not WBC nuclei since the nuclear material (DNA) is the same. The nuclear stain claimed by Inami et al., Propidium Iodide, is a commonly used vital nuclear stain that stains dead cell nuclei by permeating damaged cell membrane and intercalates into the DNA helix of any nucleus, not just an NRBC nucleus. Second, the method does not separate or distinguish the fluorescent signals of the NRBC nuclei from that of other nuclear remnants such as Howell-Jolly Bodies, Basophilic Stippling, RNA from lysed reticulocytes and reticulated platelets, and DNA from WBC and Megakaryocytic fragments. Third, the Inami et al. method requires that the sample be pretreated, off-line, using several reagents to "prep" the sample before the prepped sample/reagent solution can be loaded into the instrument.